Detection of specific oncogenic biomarkers is an important factor in choosing the proper type of targeted cancer therapy. Current cancer therapy is focused on drugs which selectively attack the cancer-causing biomarkers, inactivate molecular mechanisms responsible for cell malignancy, and deliver the toxin specifically to the malignant cells. The most prominent example of this approach is use of a monoclonal antibody (mAb) Trastuzumab antibody-drug conjugate, T-DM1 for treatment of Human Epidermal Growth Factor Receptor 2 (HER2) positive cancers. Elevated HER2 expression is associated with increased proliferation and survival of cancer cells and, thereby, contributes to poor therapy outcomes. Since the efficacy of the mAb depends on overexpression of its target on tumor cells, development of techniques to assess the receptor expression is extremely important for monitoring the efficacy of therapy and to optimize the treatment. Current clinical evaluation of HER2 expression is based on Immunohistochemistry (IHC) or Fluorescence in situ hybridization (FISH) staining of biopsied tissue. Both methodologies are ex vivo techniques and, due to tumor heterogeneity, may deliver false-positive or -negative results and can hardly be used to monitor therapy. On the other hand, molecular imaging, using HER2-specific fluorescently-labeled probes, allows assessing the status of HER2 receptors in vivo and following treatment in real time. After injection, probes like HER2-specific Affibody concentrate at the sites of interest (i.e. in the tumor). Probe accumulation at the tumor cells can occur in parallel to the delivery of some HER2 - specific drugs, such as, Trastuzumab, to the malignant tumor. As a result, HER2-specific Affibody fluorescent probes can be used to monitor HER2 expression without interference with the treatment itself, making this probe suitable for an image and treat paradigm. Besides the information that can be extracted from the serial imaging of fluorescence intensity at different time points, additional information about the tumor characteristics can be extracted from the lifetime fluorescence measurements, including binding affinity of the probe to cancer cells and environmental conditions (e.g., pH), based on local variations in the lifetimes for specially designed probes at a given site. We studied the potential of in-vivo fluorescence lifetime imaging to monitor the efficacy of treatment. In this study, we investigated the feasibility of fluorescence lifetime imaging to monitor in-vivo expression of the HER2 receptor in the breast carcinoma (mouse model) during the course of treatment. We observed considerable difference (>130 ps) between the fluorescence lifetime of HER2-specific optical probes at the tumor and contralateral site before and 7days after the last treatment with 17-DMAG (an HSP90 inhibitor), when the tumor grew back almost to its pretreatment volume. However, soon after the therapy (12 hours), when the effect of drug on HER2 degradation is maximal, this difference decreased significantly. Based on our previous findings on relationship between fluorescence lifetime and binding of HER2-specific probe to corresponding receptors, we believe that the differences between the fluorescence lifetimes at the tumor and contralateral site, observed for mice with BT-474 xenografts after treatment with 17-DMAG, results from the strong downregulation of HER2 receptors soon after the therapy (i.e., less binding sites for the HER2-specific probe) and the corresponding changes in the fraction of bound to total fluorescent probes in the tumor. Immediately after treatment, the fraction of bound to total fluorophores inside the tumor changed considerably, resulting in a noticeable increase in the average fluorescence lifetime. Subsequent tumor and HER2 expression recovery a week later caused gradual restoration of the original level of binding ratio of HER2-targeting probe in the tumor and corresponding return to pre-treatment values of the fluorescence lifetime. These results reveal that fluorescence lifetime imaging, based on evaluating the fraction of the bound and unbound fluorophores inside the tumor, can be used as an alternative in vivo imaging approach to characterize tumors, separate high and low HER2 expression tumors and monitor the efficacy of targeted therapies. Facial redness is one of the earliest described clinical features of Cushings syndrome (CS). In collaboration with NICHD (Dr. Stratakis group), we have continued the study aimed at quantifying changes of facial plethora in CS as an early assessment of cure. Cushings patients are recruited for optical imaging, before and after surgery with follow-up sessions 6 months and one year after their surgery. To date, 51 patients with CS (30F, mean age 17.814.3) have been enrolled. Among these, 38 patients had ACTH secreting pituitary tumors-Cushings disease (CD), 5 had ACTH-independent adrenocortical tumors and two had an ectopic ACTH-secreting pulmonary carcinoid. Six patients with CD were excluded from the study; two due to severe facial acne, two to fever on the day of imaging and the last two for cortisol injection on imaging day. Three of the patients required 2 consecutive surgeries, as the initial TSS was unsuccessful. Non-invasive multi-spectral near-infared imaging was performed on the right cheek of the patients before and two days or up to two weeks after surgery. Patients were defined as cured by postoperative measurements of plasma cortisol less than 3 (mcg/dl), and/or adrenocortical insufficiency for which they received replacement. Clinical data, obtained from these 45 patients, indicate that a decrease in facial plethora after surgery, as evidenced by decrease in blood volume fraction, is well correlated with cure of CS. The results will be published in the journal of clinical endocrinology & metabolism (JCEM). We are pursuing Kaposi Sarcoma (KS) studies in ongoing clinical trials under four different NCI protocols and therapeutic agents. The goal is to further evaluate diffuse multispectral imaging as a potential supplement to existing response assessment in KS, providing an early non-invasive marker of treatment efficacy. In our preliminary results, multi-spectral images of Kaposi Sarcoma skin lesions were taken over the course of treatment, and blood volume and oxygenation concentration maps were obtained through Principal Component Analysis (PCA) of the data. Corresponding images were compared with clinical and pathological assessment, provided by conventional means. In agreement with our hypothesis that successful treatment would decrease the blood volume in the lesions, the normalized standard deviation for blood volume decreased in each of the 8 patients whose lesion responded to treatment, while the normalized standard deviation for blood volume increased in 2 patients whose lesion did not respond to therapy. These initial results confirm that concentrations of oxygenated hemoglobin in the tumor can become a quantitative marker of tumor response to the therapy. We are working on designing a new multi-spectral system that has 12 wavelength imaging capabilities and will use it in a study consisting of new patients and to map more biological chromophores.